Rapid Chromosome Evolution in Recently Formed
Polyploids in Tragopogon (Asteraceae)
K. Yoong Lim1, Douglas E. Soltis2, Pamela S. Soltis3, Jennifer Tate4, Roman Matyasek5, Hana Srubarova5,
Ales Kovarik5, J. Chris Pires6, Zhiyong Xiong6, Andrew R. Leitch1*
1School of Biological and Chemical Sciences, Queen Mary College, University of London, London, United Kingdom, 2Department of Botany and the Genetics Institute,
University of Florida, Gainesville, Florida, United States of America, 3Florida Museum of Natural History and the Genetics Institute, University of Florida, Gainesville, Florida,
United States of America, 4Massey University, Institute of Molecular Biosciences, Palmerston North, New Zealand, 5Institute of Biophysics, Academy of Sciences of the
Czech Republic, Brno, Czech Republic, 6Division of Biological Sciences, Life Sciences Center, University of Missouri, Columbia, Missouri, United States of America
Background: Polyploidy, frequently termed ‘‘whole genome duplication’’, is a major force in the evolution of many
eukaryotes. Indeed, most angiosperm species have undergone at least one round of polyploidy in their evolutionary history.
Despite enormous progress in our understanding of many aspects of polyploidy, we essentially have no information about
the role of chromosome divergence in the establishment of young polyploid populations. Here we investigate synthetic
lines and natural populations of two recently and recurrently formed allotetraploids Tragopogon mirus and T. miscellus
(formed within the past 80 years) to assess the role of aberrant meiosis in generating chromosomal/genomic diversity. That
diversity is likely important in the formation, establishment and survival of polyploid populations and species.
Methodology/Principal Findings: Applications of fluorescence in situ hybridisation (FISH) to natural populations of T. mirus
and T. miscellus suggest that chromosomal rearrangements and other chromosomal changes are common in both
allotetraploids. We detected extensive chromosomal polymorphism between individuals and populations, including (i)
plants monosomic and trisomic for particular chromosomes (perhaps indicating compensatory trisomy), (ii) intergenomic
translocations and (iii) variable sizes and expression patterns of individual ribosomal DNA (rDNA) loci. We even observed
karyotypic variation among sibling plants. Significantly, translocations, chromosome loss, and meiotic irregularities,
including quadrivalent formation, were observed in synthetic (S0and S1generations) polyploid lines. Our results not only
provide a mechanism for chromosomal variation in natural populations, but also indicate that chromosomal changes occur
rapidly following polyploidisation.
Conclusions/Significance: These data shed new light on previous analyses of genome and transcriptome structures in de
novo and establishing polyploid species. Crucially our results highlight the necessity of studying karyotypes in young
(,150 years old) polyploid species and synthetic polyploids that resemble natural species. The data also provide insight
into the mechanisms that perturb inheritance patterns of genetic markers in synthetic polyploids and populations of young
natural polyploid species.
Citation: Lim KY, Soltis DE, Soltis PS, Tate J, Matyasek R et al. (2008) Rapid Chromosome Evolution in Recently Formed Polyploids in Tragopogon
(Asteraceae). PLoS ONE 3(10): e3353. doi:10.1371/journal.pone.0003353
Editor: Samuel P. Hazen, University of Massachusetts Amherst, United States of America
Received July 3, 2008; Accepted September 8, 2008; Published October 9, 2008
Copyright: ? 2008 Lim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors thank NERC, Grant Agency of the Czech Republic (521/07/0116), and the USA National Science Foundation (grants MCB-0346437, DEB-
0608268, DBI 0638536 and DBI 0501712) for support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Polyploidy has played a major role in generating angiosperm
biodiversity. Chromosome counts suggest that between 30 and
80% of angiosperm species are polyploid, while genomic studies of
selected model and crop species reveal evidence of extensive
ancient genome-wide multiplications. Indeed recent genomic
investigations indicate that most, if not all, angiosperm species
have undergone at least one genome duplication event in their
evolutionary history, and several have evidence of multiple
polyploidy-diploidisation-polyploidy cycles [1,2,3].
Angiosperm genomes are astonishingly plastic in their ability to
tolerate considerable karyotypic (e.g. chromosome number
variation, translocations), genetic (mutations, retroelement trans-
positon, deletions) and epigenetic (DNA methylation, histone
methylation/acetylation) variability. This tolerance enables poly-
ploids to form and establish and has contributed significantly to
their widespread occurrence . Large-scale genetic changes
induced by polyploidy are thought to influence the transcriptome,
metabolome and proteome, which can concomitantly alter the
phenotype and ecology of the individuals. Most of the new genetic
changes are probably maladaptive, but in a few rare instances
individuals arise that are able to outcompete the parental diploids
or colonize new niches.
There are several examples of recent speciation via polyploidy
that occurred within the last 150 years: Spartina anglica , Senecio
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cambrensis and S. eboracensis , Cardamine schulzii , Tragopogon
mirus and T. miscellus . Studies on the genetic consequences of
allopolyploidy in these de novo polyploid species reveal some
significant differences. In S. anglica allopolyploidy induced few
changes in genome structure, but there is epigenetic reprogramming
[5,9], while in Senecio [10,11] and Tragopogon  allopolyploids there
are substantial genetic changes including loss of sequence (genomic
DNA profiles) and perturbations to the transcriptome (cDNA
profiles). However, little attention has been paid to the chromosomal
content of any of these allopolyploids, and it is unknown what
mechanisms drive the observed genetic changes. For example,
genetic change could be driven by local mutation, small-scale
deletion or insertion of a particular sequence, or via major
chromosomal changes, including whole-arm transposition and
chromosome losses or duplications. Because there is little or no
understanding of chromosomal variation in these recently formed,
natural polyploids, we have embarked on a characterization of
karyotypic variation between individuals and populations of the two
Tragopogon allopolyploids from North America.
Tragopogon mirus and T. miscellus have proved to be excellent
evolutionary model systems for understanding early allopolyploid
formation. These allopolyploids are derived from three diploid
progenitors (each 2n=2x=12), T. pratensis, T. porrifolius and T.
dubius, the latter being shared by both allopolyploids (T. mirus
derived from T. dubius6T. porrifolius, 2n=4x=24; T. miscellus
derived from T. dubius6T. pratensis, 2n=4x=24, Figure 1) .
The diploid parents of both polyploids are in well separated clades
 and are not closely related based on ITS/ETS sequences,
allozymes and other genetic markers [15,16]. There are many
reports of natural F1hybrids involving these diploids and we have
also produced them in the glasshouse, but these hybrids are highly
sterile suggesting minimal homeologue pairing at meiosis . In
contrast the allopolyploids T. mirus and T. miscellus are fertile and
expanded their ranges rapidly after their initial formations, in large
part via multiple origins . The two allotetraploids now occupy
a large geographic area of eastern Washington and adjacent
Idaho, USA, and comprise many thousands of individuals in
several populations. Molecular analyses have revealed that T. mirus
has recurrently formed at least 13 times and T. miscellus possibly as
many as 21 times [13,16,18,19,20, Symonds et al. unpubl.,21],
reviewed in Soltis et al. . Furthermore, there are genetic
differences between populations of each tetraploid, most of which
likely reflect variation found in the diploids and inherited in the
polyploid populations through recurrent formation, and others of
which may have arisen through subsequent divergence of the
polyploids. Examples of the latter include the divergence of rRNA
gene copy number, sequence homogeneity and expression patterns
[22,23]. Here we show that population differences are also
reflected in substantial variability in karyotypes among individuals,
differences that appear correlated with irregular meiosis.
Genome structure of Tragopogon allotetraploids
The chromosome sizes, numbers and centromere indices are
similar between the parental diploid species, and three tandem
repeats characterized by us do not generate differences in
chromosomal distribution. For these reasons we are unable to
identify the parental origin of the chromosomes in the derived
allotetraploid species via morphology alone . We therefore
Figure 1. Tragopogon triangle with the flowers of the diploid Tragopogon species at the apices of the triangle. The flowers of the
polyploids T. miscellus and T. mirus are shown between their respective diploid parents. The synthetic polyploid described here has the same parents
as T. mirus.
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used FISH with total genomic DNA probes (called Genomic In
Situ Hybridization or GISH) to determine the genomic compo-
sition of karyotypes of T. mirus and T. miscellus individuals (Figures 2
and 3). We analysed 12 plants, nine of T. mirus from three
populations (Table 1) and three of T. miscellus, two from one
population and a third individual from a second population
(Table 2). Five of these plants, including representatives of each
tetraploid, were chosen because we had previously observed
[22,23] that they had particular 45S nuclear ribosomal DNA (45S
rDNA) compositions and expression characteristics (Table 3).
GISH labelling enabled the genomic origin of the chromosomes to
be determined by fluorescence colour: digoxingenin-labelled
genomic DNA of T. dubius labelled chromosomes of T. dubius
origin green or yellow (D-genome) and biotin-labelled genomic
DNA of either T. pratensis (to T. miscellus) or T. porrifolius (to T. mirus)
labelled the P-genome (either T. pratensis or T. porrifolius) orange or
red. However, the distinction between genomes was only possible
after electronic merger of the images because there was
considerable cross-hybridisation of probes. The homologous group
assigned to each chromosome (A–F) was determined using size,
arm ratio, position of 5S and 45S rDNA, as described in Pires et
al. , and by using DAPI bright bands that were revealed after
denaturation in some metaphases.
Previous cytogenetic analyses revealed that both T. mirus and T.
miscellus had 24 chromosomes; aneuploidy has not been previously
reported in these taxa [13,24,25]. This chromosome number was
also observed here in all but one individual of T. mirus, which had 23
chromosomes. It was assumed that GISH would partition the
chromosome sets into 12 chromosomes of T. dubius origin and 12
chromosomes of either T. pratensis or T. porrifolius origin depending
on the tetraploid being analysed. However, only four had an entirely
balanced additive karyotype of that expected from the chromosomes
of the diploid parents (Tables 1 and 2 and Figures 2 and 3). We were
surprised to observe that five of the eight T.mirus karyotypes and two
of the three T. miscellus karyoypes with 2n =24 chromosomes had
unbalanced genomic contributions. This was manifest by some
chromosomes occurring in one or three copies (monosomic and
trisomic, respectively; see Tables 1 and 2). In addition, four plants,
including individuals of both T. mirus and T. miscellus, carried
intergenomic translocations (Figures 2A, 3A, arrows).
The T. mirus individuals 2603-33A and 33B are siblings from
different flower heads of the same plant. Significantly, their
karyotypes exhibit substantial differences; 2603-33B has an
expected karyotype assuming complete additivity of parental
chromosome sets, while 2603-33A is monosomic for chromosome
Fduand trisomic for chromosome Epo.
Meiotic and mitotic aberrations in synthetic
reconstruction of T. mirus
Meiosis in newly formed, synthetic allotetraploids (S0and S1)
(see Figure 1) was examined to determine if meiotic aberrations
occurred in early allotetraploid generations and could be a source
of genomic imbalance observed in root tip metaphases. Meiotic
cells of developing anthers at diplotene were analysed (Figure 4).
In many instances regular bivalent formation occurred (Figure 4A),
although frequently bivalents overlapped (Figure 4C, D), making it
difficult to be certain if they were in fact multivalents. However, in
a number of cases resolution was sufficient to determine the
presence of quadrivalents (Figure 4B). Using GISH, the genomic
origin of the chromosomes could be resolved, although the
distinction was less clear than at metaphase. Figures 4E–F show
GISH labelling of a quadrivalent with two chromosomes of T.
dubius origin and two chromosomes of T. porrifolius origin physically
linked via chiasma in a large, twisted ring.
An analysis of root tip metaphases in 12 allotetraploids of the
subsequent S1generation revealed one plant that was 2n=23, the
restwere 2n=24, asexpected. There werecytogenetic abnormalities
in two additional plants. One of these plants (73-14) had unusually
large 45S rDNA loci occurring on both copies of chromosome Adu
(Figure 5 A, B). This plant also had an rDNA locus on chromosome
Apoof T. porrifolius origin, but the expected site on chromosome Dpo
was absent. The second plant (134-16-3) had a translocation of T.
porrifolius origin to chromosome Cdu. Since there is no missing
chromosome segment of T. porrifolius origin, it must be assumed that
this was a non-reciprocal translocation induced during the preceding
meiosis (Figure 5 C, D, E).
45S rDNA decondensation
Previous cytogenetic analyses of diploid Tragopogon species
revealed that T. dubius and T. pratensis carry a 45S rDNA locus
on chromosome Aduand Apr, respectively, while T. porrifolius has
two loci on chromosomes Apoand Dpo. To determine if these sites
were inherited in the respective allotetraploid species we used
FISH with digoxigenin-labelled probe pTa71 (yellow fluorescence,
Figures 2 and 3). In T. miscellus, 45S rDNA loci occur on
chromosomes Aduand Apr(Figure 3). At metaphase both
chromosomes carried secondary constrictions (Table 3). In T.
mirus, 45S rDNA loci occur on chromosomes Adu, Apo, and Dpo
(Figure 2). These loci were also frequently identifiable after GISH
labeling, without the use of the rDNA probe pTa71, appearing
with a brighter green (to T. dubius origin loci) or yellow
fluorescence (Figure 2). In all cells, both mitotic and meiotic, the
number of 45S loci and their parental distribution were as
expected, i.e. pairing and segregation of rDNA-carrying chromo-
somes is probably regular. This was not the case, however, for 5S
rDNA-carrying chromosomes (5S probe, red fluorescence). We
would expect T. mirus to carry 5S rDNA loci on chromosomes Adu,
Apoand Fpo. But here we observed one T. mirus individual (2602-
03-10) trisomic for chromosome Fpoand each of these chromo-
somes carried a single 5S rDNA locus (Figure 2). It was a surprise
that the chromosomes carrying the 45S rDNA were balanced
because at diplotene they were frequently in close proximity
(Figure 4 E, F), probably reflecting transcriptional activity and
nucleolar function. The absence of aberrant numbers of 45S
rDNA-carrying chromosomes at metaphase suggests that they
paired regularly at meiosis.
An analysis of 45S rDNA-carrying chromosomes in 14
diplotene nuclei of T. mirus revealed that chromosome Aduwas
always associated with the nucleolus (Figure 4E–H). Often this
chromosome was associated with a chromosome from the T.
porrifolius genome (when identifiable it was chromosome Dpo,
Figure 4G, H). Similar results were observed at metaphase
(Table 3, Figure 2), where chromosome Aduand sometimes
chromosome Dpocarried secondary constrictions (the exception is
plant 2603-33A–below). In contrast, chromosome Apowas always
condensed and unassociated with the nucleolus. Interestingly, in
two individuals of population 2602 only one of the two
homologues of chromosome Aducarried secondary constrictions.
In plant 2603-33A, there was no secondary constriction on
chromosome Aduand the 45S rDNA locus was unusually small.
Instead, chromosome Dpohad a secondary constriction. Similarly
in two synthetic T. mirus, plants 134-16-3 and 73-14, there were
secondary constrictions on Aduand Apo, with the locus on
chromosome Dpomissing in the latter individual (Figure 5 A, C).
In natural T. mirus and T. miscellus, the presence of secondary
constrictions correlated well with patterns of condensation/
decondensation at interphase (Figure 2D, E); for example,
chromosomes Apoand Dpohave no secondary constrictions in
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Figure 2. (A-C) Karyotype analyses of T. mirus from individuals in three populations (A) 2601, (B) 2603 and (C) 2602. Homeologous
chromosome group nomenclature (A-F) follows Ownbey and McCollum  and Pires et al. . Fluorochrome colours: yellow/green (FITC,
digoxigenin-labelled probes), orange/red (Cy3, biotin-labelled probes), blue (DAPI staining). Each karyotype is shown with DAPI staining, sometimes
also simultaneously labelled for 45S rDNA (yellow fluorescence) or 5S rDNA (red fluorescence), and after GISH with T. dubius (green fluorescence) and
T. porrifolius (red fluorescence) total genomic DNA probes. Monosomic or trisomic chromosomes are underlined. Note: (1) intergenomic
translocations to chromosome Cpo/du(arrows) in individuals 2601-7 and 2601-8 and Ddu/poin individual 2602-0-3-4 (A). (2) The 45S rDNA locus on the
two homologues of chromosome Aduin individuals 2602-4 and 2602-0-3-4 show different levels of decondensation, one being condensed and the
other with a secondary constriction (C). (3) A large reduction in size of the 45S rDNA locus on chromosome Aduof individual 2603-33A (#) compared
with other individuals (D). (4) No secondary constrictions of 45S rDNA loci on T. porrifolius origin chromosomes (Apo, Dpo) in individual 2603-33B. (D-E)
Root-tip interphase nuclei of T. mirus after FISH with digoxigenin-labelled pTa71 for 45S rDNA. (D) Individual 2601-4, showing four large condensed
45S rDNA loci (arrows) and (E) Individual 2602-0-3-10 with two large condensed 45S rDNA loci (arrows). Scale bar (top right) is 10 mm.
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T. mirus 2601-4 (not shown) and exhibit four sites of condensed
rDNA chromatin (Figure 2D) while in T. mirus (2602-0-3-10)
chromosome Apois without a secondary constriction (Figure 2C)
and at interphase there are two highly condensed rDNA loci
Southern blot analysis of 5S rDNA loci
Given the imbalance observed in the inheritance of individual
chromosomes, we would expect the copy number of 5S rDNA units
to vary among plants. For example, individual 2602-3-10, showing
trisomy of the Fpochromosome, had seven 5S rDNA signals instead
of the expected six (Figure 2C). On the other hand, if there was Fpo
monosomy, we might expect a reduction in gene copy numbers.
Using Southern blot hybridisation, we determined the parental T.
porrifolius/T. dubius 5S rRNA gene copy number ratio in the progeny
of a single T. mirus plant, 2602-0-3. Several progeny were studied,
including those analysed by GISH (plants 2602-0-3-10 and 2602-0-
3-4). To ascertain the parental origin of the 5S rDNA units, we
digested genomic DNA with TaqI restriction enzyme. This enzyme
takesadvantage ofa restrictionsitepresentinT.porrifolius unitsthatis
not found in T. dubius units. Consequently, after digestion the T.
little digestion of T. dubius units that remained at high molecular
weight (Figure 6). In T. mirus, the 5S rDNA probe used for Southern
hybridisation revealed signals from both parental gene families.
However, the distribution of signal intensities between genes of T.
porrifolius and genes of T. dubius origin differed between progeny. For
example, the trisomic individual 2602-0-3-10 had a T. porrifolius/T.
dubius 5S rDNA unit ratio close to 1.0, while the ‘‘normal’’ disomic
plant 2602-0-3-4 had a ratio that was 0.82. Thus, trisomy of
chromosome Fporesulted in an approximately 10% increase in the
number of 5S genes in the genome. This value is in a good
theoretical agreement with the expected increase in number of 5S
3-5,-7and-8)hada5SrDNAgene ratiocorresponding toa trisomic
genotype, suggesting meiotic aberrations were frequent in this
lineage. On the other hand, two plants (2602-0-3-2 and -3) had a
decreased number of T. porrifolius genes compared with the
expectation (plant 2602-0-3-4, with balanced chromosomal sets).
Perhaps these two individuals are monosomic for Fpo.
Effectiveness of GISH
Phylogenetic analyses of internal (ITS) and external transcribed
spacer (ETS) sequences of nuclear 45S rDNA suggest that the
diploid progenitors of both T. mirus and T. miscellus are distantly
related, with T. dubius in one major clade of Tragopogon and both T.
pratensis and the populations of T. porrifolius that served as parents
in another major clade [26,27]. Earlier allozyme studies also
suggested that the three diploid progenitors were well differenti-
ated genetically [16,28]. Likewise, comparisons of cDNA-AFLP
genetic markers between the diploid species reveal that T. pratensis
and T. dubius share only between 30-40% of markers , again
suggesting considerable genomic divergence.
Recently, Markova et al.  used GISH with genomic DNA
probes from diploid species of Silene onto metaphases of related
diploids to show that labeling strength was inversely correlated to
genetic distance, i.e. there was strongest labeling to the most
closely related species. Given these data and an apparently large
Figure 3. (A–C) Karyotype analyses of T. miscellus from two
populations (A) 2604 and (B) 2605. Homeologous chromosome
group nomenclature (A–F) follows Ownbey and McCollum  and
Pires et al. . Fluorochrome colours and probes are as in Figure 2
except that biotinylated T. pratensis genomic DNA was used in GISH
experiments. Monosomic and trisomic chromosomes are underlined.
Note in (A) that there is a large intergenomic translocation,
chromosome Adu/pr(arrow). Scale bar (top right) is 10 mm.
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genetic distance between Tragopogon diploids, we might expect
GISH to work effectively on the derived allopolyploids. Instead,
we observed that it worked rather weakly, with much cross-
hybridisation of probes, and the genomic distinction was only
resolvable after electronic merging of images. Indeed previously
we had reported that GISH had not worked at all in Tragopogon
. Our success here is due to improved quality of the electronics
on the microscope’s camera and improved facilities to electron-
ically merge images using Openlab software, but it remains an
enigma why GISH does not work more effectively in T. mirus and
rDNA inheritance and expression patterns
We expect T. miscellus to inherit two 45S rDNA loci (carried on
chromosome Adufrom T. dubius and Aprfrom T. pratensis), while T.
mirus inherits three loci (carried on chromosomes Adufrom T.
dubius and Apoand Dpofrom T. porrifolius). The observations of
only two 45S rDNA in synthetic T. mirus 73-14 (Figure 5A, B) can
be explained in one of two ways. First, the enlargement of the
rDNA locus on chromosome Aduand the locus loss on Dpo
occurred through an rDNA translocation/fusion involving the loci
on Dpoand Adu. However, given that the karyotype is balanced
and the material is the S1generation, this scenario seems unlikely
because it would suggest the union of two gametes carrying the
same abnormality, or arising via restitution of segregating
chromosomes in meiosis II. The second and alternative explana-
tion is that the rDNA locus number may reflect the situation in the
T. porrifolius parent used for the cross, although our past analyses of
T. porrifolius never revealed such a polymophism . Unfortu-
nately the precise T. porrifolius parent used in the cross is now
deceased (the plants are annuals or biennials), preventing us from
determining which of the competing hypotheses is correct.
Previously we showed that natural Tragopogon allopolyploids also
do not have fixed patterns of 45S rDNA inheritance, with some
individuals showing a balanced distribution of rDNA sequences
originating from each of the parental diploids and some showing
biased inheritance, typically with the number of rDNA units from
T. dubius being underrepresented . However, from molecular
studies it was not clear whether non-Mendelian rDNA inheritance
was caused by a decrease in copy number (elimination) or unit
replacement (e.g. via homogenisation mechanisms). In one
individual of T. mirus, 2603-33 (referred to here as 2603-33A),
Table 1. Karyotype organisation of individuals of T. mirus from three populations (localities of each population are indicated in
T. mirus populationPlant number Chromosome No. (2n)
Nature of karyotype imbalance
T. dubius originT. porrifolius origin
2601 Pullman, Washington4 24EE–
2602 Palouse, Washington0-3-423E16Cpo
2603 Rosalia, Washington 33A2416Fdu
The parental origins of the chromosomes were determined by GISH. Chromosome nomenclature of homeologous groups (A–F) followed Ownbey and McCollum 
and Pires et al. . Superscript letters indicate the genome origins of the chromosomes, du=T. dubius, po=T. porrifolius. Chromosomes carrying translocations are
indicated by naming the chromosome according to the genomic origins of the centromeres, followed by the genome origins of the translocated segments. E-indicates
chromosome set as expected from the diploid parents.
Table 2. Karyotype organisation of individuals of T. miscellus from two populations (localities of each population are indicated in
T. miscellus populationPlant number Chromosome No . (2n)
Nature of karyotype imbalance
T. dubius originT. pratensis origin
2604 Moscow, Idaho7 2436Fdu
2605 Pullman, Washington3 24EE–
The parental origins of the chromosomes were determined by GISH. Chromosome nomenclature of homeologous groups (A–F) followed Ownbey and McCollum 
and Pires et al. . Superscript letters indicate the genome origins of the chromosomes, du=T. dubius, pr=T. pratensis. Chromosomes carrying translocations are
indicated as in Table 1. E-indicates parental chromosome set as expected from the diploid parent.
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there was a considerable reduction in the number of T. dubius units
(to ,100 copies/diploid) from an expectation of ,700 units (typical
number for any given tetraploid population). An analysis of this
individual’s karyotype (Figure 2) revealed that the loss in copy
number can be accounted for by a reduction in the size of the rDNA
locus on chromosome Adu. The sibling to this plant, 2603-33B, does
not show this rDNA copy number deletion. Therefore, the locus size
reduction in 2603-33A may have arisen from a deletion event
occurring as a consequence of meiotic instability in the parent.
Despite the relatively small size (perhaps only 100 copies/
diploid of the 45S rDNA unit) of the T. dubius 45S rDNA locus, it
usually dominates rRNA expression in leaf material of T. mirus
. Even in the 2603-33A individual, with extremely reduced
numbers of 45S rRNA genes of T. dubius origin, the locus accounts
for 97% of the rRNA transcripts (see also Table 3). Nevertheless,
in root-tip metaphases a secondary constriction is observed on
chromosome Dpo, suggesting some transcription of the T. porrifolius
locus occurs in that tissue, perhaps to compensate for the reduced
45S rDNA gene copy number when there is a high demand for
ribosomes in metabolically active meristematic cells. In plant
2603-33B, with substantially higher numbers of T dubius derived
45S rRNA genes, the units of T. dubius origin are transcribed, and
those rDNA units of T. porrifolius origin are silent . This is
reflected in the lack of a secondary constriction at the rDNA locus
on chromosomes Apoand Dpoin this individual (Figure 2, Table 3).
The presence of secondary constrictions correlates strongly with
levels of decondensation at interphase and almost certainly reflects
transcriptional activity at the preceding interphase. Two individ-
uals of T. mirus (2602-4 and 2602-0-3-4) show different
decondensation of the two Aduhomologues, probably reflecting
genetic or epigenetic differentiation between the two homologues.
In population 2605 of T. miscellus, all individuals investigated show
only partial dominance in the expression of rDNA units of T.
dubius origin over those of T. pratensis origin . This is also seen
Table 3. Genomic origin of decondensed rDNA compared with previously published data from Kovarik et al.  and Matyasek et
al.  using genomic–cleaved amplified polymorphic sequence (g-CAP) and reverse transcription-cleaved amplified polymorphic
sequence (RT-CAP) to determine the genomic origin of rDNA sequences in the genome and the cDNA, respectively.
Population Figure Number
Parental origin of
genomic rDNA units
determined by g-CAPS
(% of T. dubius origin
Parental origin of
expressed rDNA units
determined by RT-CAPS,
(% of T. dubius origin
T. mirus, 2601 Pullman,
Figure 2D (interphase
Figure 2A 0-726Adu
T. mirus, 2602 Palouse,
T. mirus, 2603 Rosalia,
Figure 2C 33B26Adu
T. miscellus, 2604
T. miscellus, 2605
Ns–FISH image not shown
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Figure 4. Pollen meiosis in synthetic T. mirus. (A,B) Feulgen staining showing in (A) 12 regular bivalents and (B) one quadrivalent (arrow) and
bivalents in close association with each other and a nucleolus. (C–H). Fluorochrome colours and GISH probes are as in Figure 2. (C–D) Note
chromosome Aduin association with the nucleolus. Two bivalents, one from the T. dubius genome (yellow) and one from the T. porrifolius genome
(orange), overlapped (arrow), perhaps occurring as a multivalent. (E–F). Note the rDNA and the bivalents carrying these genes associated with the
nucleolus. A quadrivalent with two chromosomes of T. dubius origin and two of T. porrifolius origin occurring in a ring (arrow). (G–H) Note two
bivalents (Aduand Dpo) with rDNA associated with the nucleolus and the Apobivalent with condensed rDNA unassociated with the nucleolus.
N=nucleolus. Scale bar (C–H) is 10 mm.
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in the occurrence of secondary constrictions on both rDNA-
carrying chromosomes Aduand Apr(Figure 3, Table 3).
An analysis of 45S rDNA evolution in Nicotiana polyploids
indicates that parental loci are initially maintained in young
polyploids, although the sequences within a locus may be subject
to concerted evolution, and over time frames of .1 million years
individual loci are lost . In Tragopogon polyploids we do not
know which of the different karyotype variants will survive
selection and become fixed. Perhaps over longer evolutionary
timescales interlocus homogenisation and new rDNA variants will
occur and spread across all rDNA loci.
Our study shows that more than one pathway can lead to non-
Mendelian inheritance of rDNA units in allotetraploids: (i)
elimination or amplification of repeats within an array can occur
without changes in locus number (as in the case of 45S rDNA
locus in 2601-33A); (ii) a change in the number of rDNA-bearing
chromosomes (and loci) without any material change in the
number of genes at each locus (as with the 5S rDNA locus–
Figure 6); or (iii) a combination of both, although this situation was
not detected in this study.
The chromosome multiplication step of polyploidy is thought to
establish species isolation barriers between the newly formed
polyploid and its diploid parents, whilst providing a homologous
partner for each of the chromosomes. However, analyses of newly
synthesised allopolyploids reveal that early-generation individuals
are often infertile, or have highly reduced fertility, due to problems
with meiosis including irregular pairing of homologous chromo-
somes [31,32]. Selection for fecundity in synthetic polyploids is
associated with generation-by-generation increased fertility .
Nevertheless even after many thousands of years of evolution,
meiotic irregularities can still occur, as observed in Triticum aestivum
(wheat), an allohexaploid where meiotic misdivision has been
exploited in the formation of wheat aneuploid lines [33,34].
An analysis of meiosis in the newly synthesised Tragopogon
allotetraploids revealed the frequent occurrence of multivalents
(Figure 4). Such aberrant pairing patterns may result in imbalanced
chromosome contribution in subsequent generations as well as
intergenomic translocations. Both abnormalities were observed in
several of 12 synthetic (S1generation) T. mirus plants analysed at
metaphase. One plant was 2n=23 and lacked a T. dubius
chromosome and one plant carried a non-reciprocal translocation
from a T. porrifolius chromosome to chromosome Cdu(Figure 5E)
Multivalents were also observed at a low frequency in natural
populations of T. mirus and T. miscellus  and in the few F2
plants resulting from diploid F1hybrids of Tragopogon . Meiotic
abnormalities can cause unequal segregation of homeologous
chromosomes and are a likely driver of the chromosomal
imbalance between genomes observed in both Tragopogon allote-
traploids. The two sibling plants of T. mirus 2603-33 had different
karyoypes: one had a balanced karyotype of 12 chromosomes from
each parental genome (2603-33B), while the other (2603-33A) was
monosomic for chromosome Fduand trisomic for chromosome
Epo. Given these data, it is likely that the parent plant had a
balanced karyotype, and meiotic irregularities, probably arising
through multivalent formation, gave rise to the imbalanced
karyotype of plant 2603-33A.
Multivalent pairing can arise either through (1) synapsis and
recombination between homeologous chromosomes in meiosis I,
Figure 5. Analyses of root tip metaphases of synthetic T. mirus (A, B is plant 73-14; C-E is plant 134-16-3). (A, C) Metaphase (merged
images) after FISH for 45S rDNA (red) and 5S rDNA (green) and counterstained with DAPI (blue). (B) and (D) Metaphases (merged images) from (A) and
(C), respectively, after reprobing using GISH with labeled genomic DNA from T. porrifolius (pink) and T. dubius (green) and counterstained with DAPI.
In (B) note the particularly there are only four 45S rDNA sites, the sites on Adu.Chromosomes are particularly large (arrows) and the site expected on
chromosome Dpois missing. In (D) note the T. porrifolius translocation to a T. dubius chromosome (red arrow) and the satellites to both Adu
homologues are distant from the rest of the chromosome (see connecting white lines). (E) Karyotype of (D) revealing that the T. porrifolius origin
translocation is to chromosome Cduand that no T. porrifolius chromosomes lack chromatin. Scale bar is 10 mm.
PLoS ONE | www.plosone.org9October 2008 | Volume 3 | Issue 10 | e3353
or (2) synapsis between chromosomes carrying intergenomic
translocations. Four out of 12 mitotic karyotypes in plants from
natural populations had intergenomic translocations visible at
mitotic metaphase following GISH (Tables 2 and 3), and
additional smaller translocations, not resolvable by GISH, may
also be present. Nevertheless, the quadrivalent indicated in
Figure 4F (see arrow) does not show intergenomic translocations
at the resolution obtained using GISH. Our analyses of plants
from different populations, although sample sizes were small,
showed that aberrant chromosome numbers were more prevalent
for certain chromosome types. For example, chromosome A was
not involved in any of the aneuploidy evetns detected, whereas
chromosome F accounted for almost 40% of all subgenomic
chromosome imbalances. There might be several explanations for
these results. First the homology between F-type chromosomes
may be larger than between other chromosomes. It is likely that
the greater sequence and morphological similarities between
homeologous chromosomes, the more likely there will be home-
ologous and multivalent pairing. In support of this, Nicolas et al.
 observed in Brassica napus haploid hybrids that chromosomes
with the highest synteny had the highest frequency of home-
ologous pairing. Nevertheless, in T. mirus, the presence of a 5S
rDNA locus on Fpobut not on Fduindicates some divergence
between the homeologues. Secondly, all chromosomes may be
equally likely to form multivalents, but aberration in copy number
of some chromosomes, e.g. homeologous group A, may not be
favored by selection.
The surprisingly high incidence of trisomy and monosomy in
highly fertile plants with 2n=24 suggests that ‘‘compensating
trisomy’’ may be operating in natural populations of both
Tragopogon allotetraploids. Compensating trisomy was first de-
scribed by Blakeslee  to refer to a situation in which the loss of
a normal chromosome is compensated by the presence of the two
arms in new translocated associations (secondary chromosomes).
That concept was extended to include the replacement of the
primary chromosome by two tertiary chromosomes, or by a
secondary and a tertiary chromosome . Compensatory trisomy
has been reported in Datura stramonium from progeny of a plant
exposed to radium [37,38], and from crops, including from Poales
[39,40] and tomato ; all were produced experimentally .
The putative compensatory trisomy observed here may be an
example from natural plants.
The allotetraploids T. mirus and T. miscellus formed from their
diploid progenitors within the last 80 years (perhaps even within
the last 60 years) and given their biennial habit, the number of
generations to present is likely to be less than 40 . The long-
term outcome of meiotic irregularities in these species is not easily
predicted. On the one hand, the genomic imbalance will reduce
fitness and perpetuate cycles of meiotic irregularities. This may
lead to a cascade of reduced fitness, generation upon generation.
Such a phenomenon was observed in synthetic Brassica allopoly-
ploids maintained by single seed descent . In the wild, cryptic
karyotypic instability manifest by aberrant ratios of homeologous
chromosomes might ultimately lead to a slow reduction in fitness
and ultimately extinction. Perhaps this accounts for the loss of
some local populations of both T. mirus and T. miscellus  and of
some recently formed Senecio polyploid populations . Nonethe-
less, it is noteworthy that despite meiotic irregularities in the initial
synthetic S0plants and subsequent S1generations, pollen fertility
and seed set were generally high in the synthetic Tragopogon
polyploid lines (Tate et al., in prep.). In addition, selection will
favour the most fertile individuals, likely to be those with the most
regular chromosome pairing. Thus, if the population can expand
through early bottlenecks of reduced fertility, the derived
populations are likely to be more fertile with regular bivalent
pairing. Certainly in well-established allotetraploids (104–105myrs
old), e.g. of Nicotiana and Triticum, no major imbalances in
chromosome numbers or the distribution of chromosomes to
subgenomes are normally observed [44,45].
The angiosperm genome is characterised by its plasticity to
genetic change, including large-scale chromosome number
changes, aneuploidy and polyploidy . However, it may be
significant that in Tragopogon polyploids, all the imbalances in
parental chromosome dosages between individuals occurred
within a near-regular karyotype of 2n=24 (one exception at
2n=23). Given the frequency of plants showing genomic
imbalance we might expect to find more plants with unexpected
Figure 6. Genomic analysis of 5S rDNA repeats. (A) Southern blot
hybridisation of 5S rDNA probe to TaqI-digested genomic DNA of the
progeny of 2602-0-3 individual and diploid parental accessions. (B)
Quantitative representation of the parental 5S gene families deter-
mined by a phosphorimager scanning of radioactivity signals on blots
(three independent experiments).
PLoS ONE | www.plosone.org 10October 2008 | Volume 3 | Issue 10 | e3353
chromosome numbers. Perhaps there is selection against plants
that deviate from 2n=24 and against those that are nullisomic for
a particular chromosome, or perhaps our sample size was too
small to find a representative range of abnormalities.
GISH data revealed that in two out of three individuals of T.
miscellusand fourofnine individuals ofT.mirus,there is an imbalance
in the parental contribution of the chromosomes. However, the
imbalance observed resulted from monosomy or trisomy, and no
individual was nullisomic for a particular chromosome. An analysis
of 10 genes in T. miscellus using genomic and cDNA CAPS revealed
that 65% of individuals displayed losses of one of the two
homeologues and a further 5% of individuals showed silencing of
one of the two homeologues in leaves . The missing alleles were
interpreted as sequences that had been stochastically eliminated
from the T. miscellus genome. The results here may suggest that
chromosome loss or non-reciprocal translocations may contribute to
the loss of alleles, although we did not find any example of a
homozygous deletion for a chromosome segment. The differential
expression of rDNA on the Aduchromosomes of two T. mirus
individuals (Figure 2) points to epigenetic or genetic heterozygosity
between the homologous chromosomes.
Large-scale genetic changes caused by parental genome
imbalance will influence the inheritance of genetic markers, which
will in turn influence the transcriptome, proteome and metabo-
lome. Clearly genetic analyses of young or synthetic polyploids
require in-depth cytogenetic studies to assess the contribution that
chromosomal changes play in the inheritance of genetic markers.
Unfortunately in recent years that work has seldom been standard
practice. Such cytogenetic data are clearly needed even if
chromosome counts appear regular [see also 43], since a deeper
analysis of the genome and chromosome substructure can reveal
substantial chromosome dosage deviation from expectation.
Materials and Methods
Seeds of Tragopogon were collected from natural populations in
Idaho (ID) and Washington (WA) (USA) (Table 1) and planted
either in a greenhouse at the Department of Botany, University of
Florida, or in field plots at the Institute of Biophysics, Academy of
Sciences of the Czech Republic, Brno. Root tips from young,
healthy, vigorously growing plants were harvested and placed in
ice cold and saturated aqueous 2mM 8-hydroxyquinoline (Sigma-
Aldrich Company Ltd, Poole, Dorset, UK). After 60 min
incubation on ice, the roots were fixed in ethanol-acetic acid
(3:1) at room temperature overnight (several washes) and stored in
the same solution at 220uC until use. Developing anthers of 1 mm
length or less, that contain diplotene nuclei, were excised from
young buds of length 1 cm or less. Root tip and meiotic material
were fixed in freshly prepared 3:1 ethanol: glacial acetic acid for
two days. Root tip material was then transferred to 90% ethanol at
220uC for long-term storage.
Making synthetic Tragopogon allotetraploids
Descriptions of the methods used to generate synthetic polyploid
lines are given in detail in a separate paper reporting the formation
and availability of these lines for research (Tate et al. in prep).
Briefly, numerous repeated T. dubius6T. porrifolius crosses were
made, and seeds from successful crosses germinated on moist filter
paper. The chromosome number was doubled to resynthesise
polyploids that closely resemble T. mirus by placing seedlings with
fully emerged cotyledons in 0.1% or 0.25% colchicine solution
overnight. After washing with water for two-three days, the
seedlings were transferred to 2.5’’ pots with soil (and grown in the
glasshouse at the University of Florida under standard conditions).
Plants coded N197-3.132-1 and N197-4.98-1 were used for
meiotic analyses (S0generation) and root tips of selfed progeny
(S1generation) analysed in root tip mitosis, metaphases of plants
coded 134-16-3 and 73-14 are shown.
Feulgen staining of meiotic cells
For meiotic squashes, small developing inflorescences (1 cm or
less in length) were collected from the greenhouse and fixed in 3:1
(as above, for root tips). Individual anthers were then removed and
macerated in 60% glacial acetic acid, stained in aceto-orcein ,
spread under a coverslip by warming over a naked flame, and
observed using a Zeiss Photomicroscope III. At least 15 meiotic
cells per plant were scored.
Preparing cell spreads for in situ hybridisation
total genomic DNA probes (called GISH) were made using
modifications of established methods [47,48]. Briefly, root tips or
10 (Apollo Scientific Ltd, Stockport, Cheshire, UK), 0.3% (w/v)
pectolyase Y23(MP Biomedicals,Solon,Ohio,USA)and 0.3% (w/v)
drieselase (Sigma-Aldrich Company Ltd., Poole, Dorset, UK) for
28 minandtransferredto1%citratebufferfor2 h.Themeristematic
cells behind the root cap were isolated in a drop of 60% acetic acid
and squashed onto a glass slide. For meiotic preparation, fixed
anthers were dissected and meiotic cells gently dispersed into a drop
were removed following freezing with dry ice.
In situ hybridisation
Fluorescence in situ hybridisation followed standard protocols,
Figures 2–4, Leitch et al. , Figure 5, Telgmann-Rauber et al.
. Genomic DNA from T. dubius, T. pratensis, and T. porrifolius
for labelling by GISH was extracted using DNeasy Plant mini kit
(Qiagen Ltd, Crawley, West Sussex, UK) following manufacturer’s
instructions. Genomic DNA was labelled with biotin-16 dUTP
(Sigma-Aldrich Company Ltd., Poole, Dorset, UK) or digox-
igenin-11-dUTP (GE Healthcare, Chalfont St Giles, Buckingham-
shire, UK) using nick translation following standard protocols. The
probe against 5S ribosomal DNA (rDNA) was prepared by
amplifying the gene using primers described in Fulnecek et al. 
and biotin-16-dUTP-labelling protocol as described in Leitch et al.
. The probe against 45S rDNA was the clone pTa71, which
includes the 18- 26S rDNA subunit isolated from Triticum aestivum,
which was labelled with digoxignenin-11-dUTP as described in
Leitch et al. . Briefly, slides were denatured in 70% (v/v)
formamide in 26 SSC (0.3 M sodium chloride, 0.03 M sodium
citrate) at 70uC for 2 min and the hybridisation mix added
(4 mg.ml21labeled probes and 50% (v/v) formamide, 10% (w/v)
dextran sulphate, 0.1% (w/v) sodium dodecyl sulphate in 26
SSC). In situ hybridisation was carried out overnight at 37uC, after
which the slides were given a stringent wash (20% (v/v) formamide
in 0.16SSC at 42uC). Sites of probe hybridisation were detected
using 20 mg.ml21fluorescein-conjugated anti-digoxigenin IgG
(GE Healthcare, Chalfont St Giles, Buckinghamshire, UK) and
5 mg.ml21Cy3-conjugated avidin (Roche Pharmaceuticals, Lewes,
East Sussex, UK) in 46SSC containing 0.2% (v/v) Tween 20 and
5% (w/v) bovine serum albumin. Chromosomes were counter-
stained with 2 mg/ml DAPI (49,6-diamidino-2-phenylindole,
Sigma Aldrich Company Ltd., in 46 SSC) and stabilised in
Vectashield medium (Vector Laboratories Ltd, Peterborough,
UK) prior to data acquisition using either: 1) Leica DMRA2
epifluorescent microscope fitted with an Orca ER camera and
PLoS ONE | www.plosone.org11 October 2008 | Volume 3 | Issue 10 | e3353
Open Lab softwareH (Improvision, Coventry, UK) (Figures 2–4)
or; 2) Olympus BX61 epiflurescent microscope using Olympus
Microsuite 5 softwareH (Olympus America Inc, Center Valley, PA,
USA) (Figure 5). The images were analysed with Adobe
PhotoshopH version 7 and treated for colour contrast and uniform
brightness only. At least 5 mitotic or meiotic cells per plant were
scored with each probe used.
TaqI restriction enzyme digestion was carried out on genomic
DNA extracted from leaves using standard protocols  with
modifications as in . After fractionation in 1% agarose by gel
electrophoresis, the DNA was transferred to a Hybond N+
membrane (GE Healthcare, Chalfont St Giles, Buckinghamshire,
UK). The32P-labelled 5S rDNA probe was from a 120-bp XbaI/
EcoRI fragment of 5S rDNA cloned from N. tabacum . Probe
hybridisation was conducted under high-stringency conditions in
the Church-Gilbert hybridisation buffer at 65 uC overnight. The
radioactivity signals were quantified by phosphorimager scanning
(Storm, GE Healthcare, UK).
We thank Mr. R. Joseph for assistance.
Conceived and designed the experiments: DS PS AK AL. Performed the
experiments: KYL DS JT RM HS ZX AL. Analyzed the data: KYL PS
RM HS AK CP ZX AL. Contributed reagents/materials/analysis tools:
DS PS AK CP AL. Wrote the paper: DS PS JT AK CP AL.
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